Ex Vivo Manufacture of Rapamycin-Resistant Human Th1/Tc1 Cells.

Previously, we found that ex vivo usage of rapamycin and a type I polarizing cytokine could be utilized to generate murine Th1/Tc1 cells that persisted in vivo and mediated increased GVHD. The type I cytokine profile and increased in vivo persistence of this population may prove useful in autologous transplant settings. In recent experiments using culture flasks and polarization with IFN-α, we found that human T cells similarly can adopt a Th1/Tc1 phenotype in rapamycin; furthermore, such human T cells have increased in vivo persistence when transferred into immune-deficient murine hosts. In this project, our goal was to evaluate whether human Th1/Tc1 cell generation in rapamycin varied depending on use of culture flasks or more clinically relevant, closed system polyolefin culture bags.

Human lymphocytes were collected by steady-state apheresis and subsequent counter flow centrifugal elutriation. CD3+ T cells were purified and co-stimulated using anti-CD3/anti-CD28 tosyl-activated P450 magnetic beads. T cells were cultured under four conditions: recombinant human (rh) IL-2 alone; rhIL-2 plus rapamycin; rhIL-2 plus rhIFN-α; or rhIL-2 plus rhIFN-α plus rapamycin. After 6 days of culture in flasks or bags, cells were harvested and co-stimulated; cytokine production was measured by Luminex assay, and cell surface markers were assessed using flow cytometry. Cells were further characterized using an in vivo xenogeneic cytokine storm model of GVHD (x-GVHD). Specifically, immune deficient Rag2/γc knockout mice were irradiated, injected with ex vivo expanded T cells, and challenged with lipopolysaccharide (LPS) injection on day 6 after adoptive transfer to induce lethal levels of TNF-α.

Relative to T cells expanded in only IL-2, further addition of IFN-α yielded T cells with increased secretion of IFN-γ (33.5 vs. 5.7 ng/ml, p=0.01) and reduced secretion of IL-4 (0.6 vs.18.7 pg/ml, p=0.02). Inclusion of both IFN-α and rapamycin also yielded cells with preferential secretion of IFN-γ relative to IL-4; IFN-γ and IL-4 secretion values did not vary significantly between flask and bag conditions. However, relative to flask expanded T cells, Th1/Tc1 cells expanded in bags had increased co-expression of T central memory molecules CD62L and CCR7 (median percentage of co-expression increased from 20% to 40%, p<0.05). Because of the increased type I polarity of T cells expanded in bags in the presence of IFN-α, we hypothesized that such cells would mediate increased x-GVHD relative to T cells expanded in bags in the absence of IFN-α. However, contrary to our hypothesis, lethality after LPS challenge was actually reduced in recipients of IFN-α exposed human T cells (p<0.05). Given these results, we reasoned that IFN-α might also induce production of the counter-regulatory cytokine, IL-10. Indeed, relative to T cells expanded in IL-2 alone, further addition of IFN-α increased IL-10 secretion from 679 to 5982 pg/ml (without rapamycin; p<0.05) and from 85 to 2302 pg/ml (with rapamycin; p<0.05). Intra-cellular flow cytometry demonstrated that Th1/Tc1 cell co-expression of IFN-γ and IL-10 occurred at the single cell level.

Ex vivo manufacture of human Th1/Tc1 cells in clinical grade polyolefin bags resulted in T cells with a differential cytokine phenotype relative to T cells cultured in flasks. Using bags, addition of IFN-α to culture yielded increased T cell capacity to secrete both IFN-γ and IL-10; this phenotype occurred independent of whether the culture also included rapamycin. The observed co-expression of IFN-γ and IL-10 supports a model whereby human Th1/Tc1 cells can simultaneously express pro- and anti-inflammatory cytokines. Th1/Tc1 cell co-expression of IFN-γ and IL-10 appeared to moderate inflammation in vivo because recipients of such bag-cultured T cells had reduced lethal x-GVHD relative to recipients of flask-cultured T cells. As such, we conclude that type of culture vessel (bag vs. flask), presence of a polarizing cytokine (IFN-α), and level of IL-10 secretion are important variables to consider for the ex vivo manufacture of human Th1/Tc1 cells.

No relevant conflicts of interest to declare.